U.S. patent application number 15/573960 was filed with the patent office on 2018-10-11 for method and system for cell cultivation.
The applicant listed for this patent is GE HEALTHCARE BIO-SCIENCES AB. Invention is credited to Bjorn Johan Lundgren, Emmanuel Macedo, Ann-Christin Magnusson, Lena Maria Sandberg.
Application Number | 20180291326 15/573960 |
Document ID | / |
Family ID | 53677362 |
Filed Date | 2018-10-11 |
United States Patent
Application |
20180291326 |
Kind Code |
A1 |
Lundgren; Bjorn Johan ; et
al. |
October 11, 2018 |
Method and System for Cell Cultivation
Abstract
The present invention relates to a method and system for aseptic
material transfer from a storage container to a bioreactor. More
precisely the invention relates to a method and system for
aseptically transferring dry material, such as microcarriers for
cell cultivation, to a bioreactor, comprising transferring
microcarriers from a first container housing said microcarriers to
a bioreactor for cell cultivation via r transfer tubing connecting
the first container to the bioreactor, wherein the transfer is
accomplished with pressurized air or gas supplied to the
container.
Inventors: |
Lundgren; Bjorn Johan;
(Uppsala, SE) ; Macedo; Emmanuel; (Uppsala,
SE) ; Magnusson; Ann-Christin; (Uppsala, SE) ;
Sandberg; Lena Maria; (Uppsala, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GE HEALTHCARE BIO-SCIENCES AB |
UPPSALA |
|
SE |
|
|
Family ID: |
53677362 |
Appl. No.: |
15/573960 |
Filed: |
May 16, 2016 |
PCT Filed: |
May 16, 2016 |
PCT NO: |
PCT/EP2016/060937 |
371 Date: |
November 14, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 29/14 20130101;
C12M 25/16 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12M 1/12 20060101 C12M001/12 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 2015 |
GB |
1509193.7 |
Claims
1. A method for aseptically transferring dry material for cell
cultivation to a bioreactor, comprising transferring dry material
from a first container housing said dry material to a bioreactor
for cell cultivation via transfer tubing connecting the first
container to the bioreactor, wherein the transfer is accomplished
with pressurized air or gas supplied to the container via a vent
tubing including a dip tubing inserted into the container.
2. Method according to claim 1, comprising transferring a portion
of dry material from a supply of dry material provided in a second
container to the first container via transfer tubing between the
containers with pressurized air or gas supplied to the container
via a vent tubing including a dip tubing inserted into the
container, and then transferring said portion of dry material from
the first container to the bioreactor with pressurized air or
gas.
3. Method according to claim 2, wherein at least two portions of
dry material are separately transferred from the supply of dry
material in the second container to the first container and then
each portion is separately transferred to the bioreactor.
4. Method according to claim 2, wherein said portion of dry
material transferred to the first container is weighed in the first
container before being transferred to the bioreactor.
5. Method according to claim 2, comprising turning the container
upside down before transfer of the dry material with pressurized
air or gas, wherein the dry material are at the bottom of the
container and the dip tubing reaches above the level of the dry
material.
6. Method according to claim 2, wherein the dry material is
selected from microcarriers, dried media, powder or salts.
7. Method according to claim 6, wherein the dry material is
microcarriers for cell cultivation.
8. A transfer system comprising one or more rigid containers
holding dry material; one or more flexible transfer tubings for
transferring dry material from the container to a bioreactor or to
a first container from a second container; one or more flexible
vent tubings including a dip tubing inserted in the container and
connected to a source of pressurized air or gas.
9. Transfer system according to claim 8, wherein the containers are
bottle shaped and provided with an upper ported cap in which the
tubings are attached to the containers.
10. Transfer system according to claim 8, wherein the container,
which is connected to the bioreactor, is placed on a scale.
11. Transfer system according to claim 8, wherein the tubings are
provided with valves and connectors.
12. Transfer system according to claim 8, wherein the dry material
is selected from microcarriers, dried media, powder or salts.
13. Transfer system according to claim 8, which is sterilized,
preferably by gamma radiation.
14. Transfer system according to claim 13, wherein the dry material
is microcarriers for cell cultivation.
15. Use of a transfer system according to claim 14 in a cell
culture process, wherein microcarriers are provided to the
bioreactor at any desired time in the process.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method and system for
aseptic material transfer from a storage container to a bioreactor.
More precisely the invention relates to a method and system for
aseptically transferring dry material, such as microcarriers, for
cell cultivation to a bioreactor, comprising transferring dry
material from a first container housing said material to a
bioreactor for cell cultivation via transfer tubing connecting the
first container to the bioreactor, wherein the transfer is
accomplished with pressurized air or gas supplied to the
container.
BACKGROUND OF THE INVENTION
[0002] Cell culture techniques have become vital to the study of
animal cell structure, function and differentiation and for the
production of many important biological materials, such as virus
vaccines, enzymes, hormones, antibodies, interferon's, nucleic
acids and virus vectors for gene therapy. Furthermore, cell culture
and cell expansion is a very important step in cell therapy. For
many cell culture approaches it is desired to expand a cell culture
from a small to a large cell population.
[0003] For cell culture it is conventional to grow the cells on a
cell adhering surface since most mammalian cells and certain other
cells are anchorage-dependent to be able to grow. Conventional cell
culture in tissue culture treated bottles or other vials give a
limited yield of anchorage-dependent cells due to the small surface
areas available.
[0004] Microcarrier culture introduces new possibilities and for
the first time makes possible the practical high yield culture of
anchorage-dependent cells. In microcarrier culture cells grow as
monolayers on the surface of small spheres which are usually
suspended in culture medium by gentle stirring. By using
microcarriers in simple suspension culture systems it is possible
to achieve yields of several million cells per milliliter and the
systems are easily scalable.
[0005] In the microcarrier approach, cell culture is usually
performed with beads in a spinner flask or beads packed in columns
(perfusion culture). More recently, cell culture with microcarriers
has been performed in disposable bioreactor, such as flexible bags.
The microcarriers are for example dextran, cellulose or
polyethylene based products.
[0006] In order to perform cell culture it is necessary to have
sterile cell culture materials, such as drya culture media and
microcarriers. The traditional way to sterilize microcarriers is
via autoclaving outside of the bioreactor or inside the bioreactor
via steam sterilization. With the introduction of single use
bioreactors, it is no longer possible to perform the sterilization
via steam in the bioreactor. Therefore it is necessary to provide
pre-sterilized microcarriers which can be introduced into the
bioreactor asceptically without compromising the sterility.
[0007] US2014/0196791 describes a single use bioreactor comprising
a microcarrier container and a bioreactor vessel, wherein the
microcarriers are transferred to the vessel by gravity or flushing
with a fluid. Transfer with gravity will not be complete, i.e. some
of the microcarriers will stick to the walls of the container and
not be transferred to the bioreactor vessel. To improve this,
flushing of the container is suggested with for example cell
cultivation medium.
[0008] In spite of the many bioreactors known in the art there is
still a need of improved methods and systems for aseptic transfer
of dry materials, such as microcarrires, to bioreactors.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method and system for
aseptic transferal of dry materials, preferably microcarriers, to
bioreactors. The system is useful with any type or size of
bioreactor and is especially useful for large scale, single use
bioreactors.
[0010] Thus, in a first aspect the invention relates to a method
for aseptically transferring dry dry material for cell cultivation
to a bioreactor, comprising transferring dry material from a first
container housing said material to a bioreactor for cell
cultivation via transfer tubing connecting the first container) to
the bioreactor, wherein the transfer is accomplished with
pressurized air or gas supplied to the container via a vent tubing
including a dip tubing inserted into the container. The dip tubing
prevents the whirling around of the dry material during
introduction of the air or gas flow.
[0011] In one embodiment, the method comprises dosing of dry
material from multiple containers by transferring a portion of dry
material from a supply of dry material provided in a second
container to the first container via transfer tubing between the
containers. The transfer is performed with pressurized air or gas
supplied to the second container via a vent tubing including a dip
tubing inserted into the second container. Thereafter said portion
of dry material which is now in the first container is transferred
to the bioreactor with pressurized air or gas.
[0012] A number of portions but preferably at least two portions of
dry material may be separately transferred from the second
container to the first container and then each portion is
separately transferred to the bioreactor. In this way it is not
necessary to connect a new container to the bioreactor when more
dry material is needed for the cell cultivation. This will minimize
the risk of contamination of the cell culture further.
[0013] In a further embodiment, said portion of dry material
transferred to the first container is weighed in the first
container before being transferred to the bioreactor. This will
guarantee that an exact amount of dry material is transferred to
the bioreactor. Each portion may comprise for example, 1 g to 10's
of kilos of dry materials giving a very flexible dosing range,
depending on the size of the bioreactor and of the containers.
[0014] Preferably the container is turned upside down before
transfer of the dry material with pressurized air or gas, wherein
the dry material are at the bottom of the container and the dip
tubing reaches above the level of the dry material.
[0015] The dry material may for example be microcarriers, dry
media, such as dried or powdered cell culture media, or any other
powders or salts.
[0016] In a second aspect, the invention relates to a transfer
system comprising one or more rigid containers holding dry
material; one or more flexible transfer tubings for transferring
any dry material, such as microcarriers, from the container to a
bioreactor or to a first container from a second container; one or
more flexible vent tubings including a dip tubing inserted in the
container and connected to a source of pressurized air or gas.
Preferably the tubings are provided with valves and connectors to
connect the tubings to the bioreactor and/or further containers.
Preferably the vent tubing is provided with a sterile vent filter.
Alternatively, sterile gas or air is used. Preferably the
containers are bottle shaped and provided with an upper ported cap
in which the tubings are attached to the containers. The containers
or bottles and their contents of dry material may have any suitable
size depending on the bioreactor size and the needs of the cell
cultivation. Examples of container sizes are from 100 mL to 5 L and
examples of bioreactor sizes are from 1 L to 10000 L. When more
than one container is used in the transfer system, they may be of
the same or different size.
[0017] Preferably the transfer system is sterilized by any type of
sterlilization, most preferably by gamma radiation.
[0018] In one embodiment the container, which is connected to the
bioreactor, may be placed on a scale for weighing the amount of dry
material transferred to the bioreactors which will be described
more closely in the detailed section below.
[0019] In a third aspect the invention relates to use of the above
transfer system in a cell culture process, wherein microcarriers
are provided to the bioreactor at any desired time in the process.
The microcarriers are provided at the beginning of the process and
at any time and in any number thereafter before the end of the
process. In one embodiment the transfer system is preferably
connected to the bioreactor during the whole process, especially
when there is a need of adding more microcarriers during the
process.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a schematic view of the transfer system of the
invention in a position ready to use, i.e. the container has been
turned upside down for transfer of dry material from a first
container into a bioreactor;
[0021] FIG. 2 is a schematic view of the transfer system of the
invention in a position for transferring a portion of the total
amount of dry material from a first container to a second container
wherein the second container is placed on a scale and the transfer
is continued until a desired weight of the dry material is obtained
in the second container; and
[0022] FIG. 3 shows a position of the transfer system in which the
second container of FIG. 2 has been turned upside down and the
transfer system is ready for use to transfer dry material from the
second container to the bioreactor via pressurized air or gas.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The transfer system of the invention and its method of use
will now be described more closely in association with the
accompanying drawings. Although the method and transfer system is
suitable for transferring any type of dry material the detailed
description below is described in connection with microcarriers for
cell cultivation as the dry material.
[0024] The present invention relates to a method and transfer
systems for aseptic transfer of dry material, such as
microcarriers, to bioreactors. The transfer system is a separate
unit from the bioreactor and can be used with any type of
bioreactor in any scale. Microcarriers used in the invention are
kept sterile in specially designed containers enabling direct
transfer of microcarrier beads into a bioreactor in an aseptic
manner. This will considerably improve the current procedure as it
avoids the tedious and time consuming preparative sterilization
practiced in the pharmaceutical industry, in which microcarrier
beads are swollen, washed with buffer and autoclaved before being
added to the cell culture vessel (bioreactor), and then washed with
cell culture media.
[0025] With reference to FIG. 1, the transfer system comprises dry
microcarriers 1 housed in a container 2 which preferably is shaped
like a bottle having a ported cap 7 through which transfer tubing 3
and vent tubing 4 is connected to the container 2. In FIG. 1 the
container or bottle 2 is shown in an upside sown position wherein
the microcarriers 1 will be located at the bottom of the container
closest to the cap 7 and the dip tube 5 will reach above the level
of the microcarriers to enable inflow of pressurized air or gas
into this space of the container which effectively will transfer
the microcarriers through the transfer lines 3 to a bioreactor (not
shown) or from a further container 2A to the container 2 as shown
in FIG. 2.
[0026] FIG. 2 shows a preferred embodiment in which dry
microcarriers 1A are transferred from a second container 2A, which
is positioned upside-down, to the first container 2 by connecting a
source of pressurized air or gas to a vent filter 6A. In this
embodiment not all microcarriers from the second container 2A are
transferred to the container 2 at the same time. On the contrary,
separate portions or doses of the microcarriers 1A are transferred
to the bioreactor via container 2 according to the needs of the
cell culture procedure. To ensure that a correct amount of
microcarriers are transferred from container 2A to container 2 and
then to the bioreactor, the container 2 is place on a scale 8. This
avoids the needs of connecting a new container 2 to the bioreactor
every time there is a need of new additions of microcarriers to the
cell culture in the bioreactor.
[0027] The microcarriers 1, 1A used in the invention are preferably
Cytodex.TM. (GE Healthcare Bio-Sciences AB) microcarriers but any
type of microcarriers may be used.
[0028] FIG. 3 shows the positioning of containers 2, 2A following
completed transfer of a desired portion of microcarriers from
container 2A to container 2. In this position the container 2A has
been brought back into its upright position and instead container 2
is turned upside down. When pressurized air or gas is supplied to
the container 2 the weighed microcarriers in container 2 will be
transferred from container 2 to the bioreactor (not shown). This
procedure, i.e. the filling of microcarriers from container 2A to
container 2 according to FIG. 2, and then the transfer to the
bioreactor according to FIG. 3, may be repeated an optional number
of times.
[0029] In the method of the invention the sterile containers 2, 2A
are readily connected to the bioreactors via tubing using an
aseptic connector or a welding connection, to provide a connection
for transfer of the microcarrier beads into the bioreactor.
Transfer will be achieved by using a slight over pressure of
sterile air or nitrogen introduced in the container via a dedicated
port, after turning the container upside down.
[0030] All the materials used for the transfer system, directly in
contact with sterile microcarrier beads, will meet USP class VI
requirements (biological test for plastics). The transfer system of
the invention including microcarrier beads preferably withstands a
maximum gamma irradiation dose of at least 40 KGy.
[0031] The microcarriers used in the invention preferably are
provided in rigid plastic container, such as PET containers. Each
container is preferably equipped with a suitable 3-ported cap 7 and
two transfer ports fitted with a tubing 3, 3A of suitable length,
such as a TPE (thermoplastic elastomer) tubing. In one embodiment
one transfer tube may be equipped at its remaining end with a
ReadyMate.TM. aseptic connector (for ReadyMate aseptic connection)
whereas the second one will be double sealed for subsequent welding
connection. In another embodiment both transfer lines are double
sealed. The third port will be equipped with a vent tubing 4, 4A,
such as a silicon vent tubing, fitted with a vent filter, such as a
0.2 .mu.m vent filter, for introduction of slightly compressed air
or nitrogen (max. 0.5 bar) for the transfer of microcarriers into
the bioreactor.
[0032] The two transfer ports 3, 3A will allow simultaneous
multiple aseptic connections, or subsequent aseptic connections, by
for example welding, to other bioreactors should a container not be
completely emptied after a previous run. The 3-ported cap is also
equipped with an internal spigot which is directly connected to the
external vent port. This spigot will be fitted with a polymeric dip
tube to bring the slightly compressed air or nitrogen over the
level of the microcarrires (once the container is turned upside
down) and press thereby material out of the container without any
major turbulence (FIG. 2-3).
[0033] The container system of the invention can be easily
connected to bioreactors via any connection, such as a ReadyMate or
by only welding aseptic connection. After turning the container
upside down, connection is opened and microcarriers are transferred
by introducing a slight overpressure of air or inert gas (FIG.
2).
[0034] If required, aseptic connection can also be achieved by
welding, providing that the container and the bioreactor are having
the same kind of tubing with matching diameters.
[0035] Should the contents of microcarriers in one container not be
enough, then it will be possible to transfer in an aseptic manner
extra material to the bioreactor following the procedure described
below, see FIG. 2-3: [0036] Place the emptied first container 2 on
a balance. [0037] Connect, for example by welding, a tubing 3A on a
second container 2A filled with a supply of microcarriers to the
remaining tubing 3 available on the emptied container 2 (FIG. 2).
[0038] Turn the second container 2A upside down and transfer/weigh
the desired amount of microcarriers from the container 2A to the
container 2 using a slight over pressure of air or nitrogen
introduced via the sterile vent tubing on the new container or
pack. [0039] Once the required amount of material has been weighed,
connection between the new container 2A and the
bioreactor-connected container 2 is closed, and the latter is
turned upside down. The newly weighed material is then transferred
into the bioreactor using a slight over pressure of air or inert
gas introduced via vent tubing on the container directly connected
to the bioreactor (FIG. 3).
[0040] If welding is the selected method to connect the
microcarrier container to a bioreactor, then the bioreactor should
be provided with the same kind of tubing of the same diameter as
the microcarrier container. If connection between the microcarrier
container and bioreactor is to be achieved via ReadyMate.TM.
aseptic connector, then there should be a ReadyMate.TM. connector
available on bioreactor tubing.
[0041] Both transfer and vent tubing are equipped with valves, such
as pinch valves, allowing selective opening/closure under transfer
operations. The valves are preferably made of a material
withstanding gamma irradiation.
[0042] The container system of the invention will be filled with
microcarriers, preferably Cytodex.TM. microcarriers, in an
environment with controlled bioburden. After filling and closing
with the ported caps, the container system will be enclosed/sealed
in a double plastic film protection (secondary/tertiary packaging)
and packed in a cardboard box. The card board box with its contents
is then gamma sterilized and the shelf life will be at least 2
years at 30.degree. C.
* * * * *